Radioimmunoassay (RIA)

The technique of
radioimmunoassay has
revolutionized research
and clinical practice in
many areas, e.g.,
• blood banking
• diagnosis
of allergies
• endocrinology
The technique was
introduced in 1960 by
Berson and Yalow as an
assay for the
concentration
of insulin in plasma. It
represented the first time
that hormone levels in the
blood could be detected
by an in vitro assay.
The Technique
• A mixture is
prepared of
○ radioactive
antigen
 Because of the ease with which iodine atoms can be introduced
into tyrosine residues in a protein, the radioactiveisotopes 125I
or 131I are often used.
○ antibodies ("First" antibody) against that antigen.
• Known amounts of unlabeled ("cold") antigen are added to samples of the
mixture. These compete for the binding sites of the antibodies.
• At increasing concentrations of unlabeled antigen, an increasing amount of
radioactive antigen is displaced from the antibody molecules.
• The antibody-bound antigen is separated (see below) from the free antigen
in the supernatant fluid, and
• the radioactivity of each is measured.
• From these data, a standard binding curve, like this one shown in red, can be
drawn.
 • The samples to be assayed (the unknowns) are run in parallel.
• After determining the ratio of bound to free antigen ("cpm Bound/cpm
Free") in each unknown, the antigen concentrations can be read directly
from the standard curve (as shown above).

Separating Bound from Free Antigen

There are several ways of doing this.
• Precipitate the antigen-antibody complexes by adding a "second" antibody
directed against the first. For example, if a rabbit IgG is used to bind the
antigen, the complex can be precipitated by adding an antirabbit-IgG
antiserum (e.g., raised by immunizing a goat with rabbit IgG). This is the
method shown in the diagram above.
• The antigen-specific antibodies can be coupled to the inner walls of a test
tube [View another example]. After incubation,
○ the contents ("free") are removed;
○ the tube is washed ("bound"), and
○ the radioactive of both is measured.
• The antigen-specific antibodies can be coupled to particles, like Sephadex.
Centrifugation of the reaction mixture separates
○ the bound counts (in the pellet) from
○ the free counts in the supernatant fluid.
Radioimmunoassay is widely-used because of its great sensitivity. Using
antibodies of high affinity (K0 = 108–1011 M−1), it is possible to detect a few
picograms (10−12 g) of antigen in the tube.
[Link to page that discusses antibody affinity]
The greater the specificity of the antiserum, the greater the specificity of the assay.
Link to an illustration of antibody specificity demonstrated by radioimmunoassay.
The main drawbacks to radioimmunoassay are the expense and hazards of
preparing and handling the radioactive antigen.
• Both 125I or 131I emit gamma radiation that requires special counting
equipment;
• The body concentrates iodine atoms — radioactive or not — in the thyroid
gland where they are incorporated in thyroxine (T4).
The enzyme-linked immunosorbent assay (ELISA) has many of the advantages (e.g.,
sensitivity, ease of handling multiple samples) without the disadvantages of dealing with
radioactivity. Link to a description of ELISA
Despite these drawbacks, RIA has become a major tool in the clinical laboratory
where it is used to assay
• plasma levels of:
○ most of our hormones;
○ digitoxin or digoxin in patients receiving these drugs;
○ certain abused drugs
• for the presence of hepatitis B surface antigen (HBsAg) in donated blood;
• anti-DNA antibodies in systemic lupus erythematosus (SLE).
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9 March 2011